Structural Similarities Among RNA Virus Class I Fusion Proteins.
Hemagglutinin 2 (HA2) of influenza virus, an orthomyxovirus, is the prototypic RNA virus Class I fusion protein and contains an amino terminal hydrophobic domain, referred to as the fusion peptide, that is exposed during cleavage of the hemagglutinin precursor protein. The membrane fusion proteins of RNA viruses from several diverse families, including arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses, share several common structural features with HA2 and have been referred to as Class I viral fusion proteins.
It has been observed that the fusion protein of HIV-1, the transmembrane glycoprotein and other retroviral transmembrane proteins, like those of orthomyxoviruses and paramyxoviruses, possess a hydrophobic fusion peptide domain exposed during cleavage of a precursor (gp160) (Gallaher, 1987; Gonzalez-Scarano et al., 1987). Based on these similarities and computer algorithms that predict protein configurations, it has been suggested (Gallaher et al., 1989) that the external portion (ectodomain, amino terminus) of HIV-1 transmembrane protein and the transmembrane proteins of other retroviruses, all could fit the scaffold of HA2 structure as determined by x-ray crystallography (Wilson, Skehel, and Wiley, 1981).
Based on these observations, it was predicted that retroviral transmembrane proteins contain several structural features in addition to the fusion peptide in common with the known structure of HA2, including an extended amino terminal helix (N-helix, usually a “heptad repeat” or “leucine zipper”), a carboxyl terminal helix (C-helix), and an aromatic motif proximal to the transmembrane domain. The presence of at least four out of these five domains defines a viral envelope protein as a Class I fusion protein. This retroviral transmembrane protein model was subsequently confirmed by structural determinations and mutational analyses (Chan et al., 1997; Kowalski et al., 1991; Weissenhorn et al., 1997). Common structural motifs are present not only in orthomyxovirus and retrovirus fusion proteins, but also in those of paramyxoviruses, filoviruses (such as Ebola virus, EboV) (Gallaher, 1996) and arenaviruses (Gallaher, DiSimone, and Buchmeier, 2001). The Gallaher structural model of the EboV fusion protein (GP2) has also been confirmed by x-ray crystallographic methods (Malashkevich et al., 1999; Weissenhorn et al., 1998).
FIG. 1 shows the five, previously-described, domains of the fusion proteins of the six families of Type I viruses. The fusion proteins originate in a hydrophobic fusion peptide, terminate in an anchor peptide, and incorporate an extended amino terminal alpha-helix (N-helix, usually a “heptad repeat” or “leucine zipper”), a carboxyl terminal alpha-helix (C-helix) (Carr and Kim, 1993; Suarez et al., 2000; Wilson, Skehel, and Wiley, 1981), and sometimes an aromatic motif proximal to the virion envelope. Also shown is the sixth domain, the fusion initiation region (FIR), discovered by the present inventors.
Fusion Inhibition in Type I Viruses.
Previous attempts by the present inventors (Garry) and others to design peptides and peptide mimics, antibodies, and other factors that inhibit fusion in Type I viruses have focused on the fusion peptide, the N-helix, and the C-helix of the fusion proteins. In the case of fusion peptides, analogs of the orthomyxoviruses and paramyxoviruses (Richardson, Scheid, and Choppin, 1980) and HIV-1 fusion peptide domains (Gallaher et al., 1992; Owens et al., 1990; Silburn et al., 1998) have been found to block viral infection, presumably by forming inactive heteroaggregates. Peptides corresponding to portions of the N-helix and C-helix have also been found to be effective in inhibiting viral infection both in vitro and in vivo. For example, a 17-amino-acid peptide corresponding to the carboxy-terminal portion of the N-helix of the HIV-1 fusion protein, defined as the CS3 region, blocked HIV infection (Qureshi et al., 1990). In addition, other N-helix and C-helix inhibitory peptides were developed based on the fusion protein structural model (Wild, Greenwell, and Matthews, 1993; Wild et al., 1992), including the C-helix anti-HIV-1 peptidic drug DP178 (T-20 or FUZEON®). DP178 overlaps the C-helix and the aromatic anchor-proximal domain and inhibits HIV-1 virion: cell fusion at very low concentrations (50% inhibition at 1.7 nM) achievable in vivo following injection. In a clinical trial, 100 mg/day of DP178 caused an approximately 100-fold reduction in plasma HIV-1 load of infected individuals (Kilby et al., 1998). This result has greatly motivated the search for other HIV-1 inhibitory peptides based on transmembrane protein structure (Pozniak, 2001; Sodroski, 1999). Peptidic inhibitors of paramyxoviruses have also been shown to inhibit viral replication (Lambert et al., 1996; Young et al., 1999). Studies by Watanabe and coworkers suggest that a similar approach of targeting the N-helix and the C-helix of EboV GP2 may also lead to useful inhibitors (Watanabe et al., 2000). Neutralizing antibodies directed against portions of the fusion protein domains have also been shown to inhibit virion:cell fusion.
Observations in HIV-1.
A great deal of study has been devoted to fusion inhibition in human immunodeficiency virus HIV-1, one of the Type I RNA viruses. Bolognesi et al. (U.S. Pat. No. 5,464,933) and the present inventors (Garry, U.S. Pat. No. 5,567,805) teach that HIV-mediated cell killing can be inhibited by introducing peptides that bind to portions of the transmembrane fusion protein of the HIV-1 virion. The Bolognesi DP178 binding region, labeled FUZEON® in FIG. 7, lies primarily on the C-helix and is outside what is described in the present application the fusion initiation region (FIR). Bolognesi demonstrates inhibition but teaches no method of inhibition. The present inventors (Garry) previously demonstrated inhibition at the CS3 region of HIV-1™, labeled CS3 in FIG. 7, but identified no method of inhibition, suggesting only that CS3: CS3-receptor interaction is inhibited. The unexpected discovery of the FIR by the present inventors (as currently described herein) and the fact that the CS3 sequences lie within the FIR indicates that the CS3: CS3-receptor binding described in U.S. Pat. No. 5,567,805 is in fact binding that occurs between the CS3 portion of the FIR and portions of the cell membrane for which the CS3 portion of the FIR has an affinity. In addition, although Melikyan, Watanabe, Bewley, and others have described fusion inhibition with introduced peptides, they have not explained the mechanisms through which the inhibition occurs. Correspondingly, the location of the FUZEON® peptide is distant from the FIR, which strongly suggests that other elements of the fusion process operate in the FUZEON® region.
In view of the foregoing, it is clear that there exists a need in the art for a more effective means for identifying those regions of viruses that are involved in the infection process and for compositions effective for preventing or inhibiting viral infection. The invention described and disclosed herein provides an effective solution to these needs.